Methods for inhibiting MRP1

ABSTRACT

The present invention relates to a compound of formula (I) which is useful for inhibiting neoplasms where the resistance is conferred in part or in total by MRP1.

This application is a 371 of PCT/US99/07615 filed Apr. 7, 1999 whichclaims priority from Provisional Application No. 60/081,088 filed Apr.8, 1998.

Along with surgery and radiotherapy, chemotherapy continues to be aneffective therapy for many cancers. In fact, several types of cancer arenow considered to be curable by chemotherapy and include Hodgkin'sdisease, large cell lymphoma, acute lymphocytic leukemia, testicularcancer and early stage breast cancer. Other cancers such as ovariancancer, small cell lung and advanced breast cancer, while not yetcurable, are exhibiting positive response to combination chemotherapy.

One of the most important unsolved problems in cancer treatment is drugresistance. After selection for resistance to a single cytotoxic drug,cells may become cross resistant to a whole range of drugs withdifferent structures and cellular targets, e.g., alkylating agents,antimetabolites, hormones, platinum-containing drugs, and naturalproducts. This phenomenon is known as multidrug resistance (MDR). Insome types of cells, this resistance is inherent, while in others, suchas small cell lung cancer, it is usually acquired.

Such resistance is known to be multifactorial and is conferred by atleast two proteins: the 170 kDa P-glycoprotein (MDR1) and the morerecently identified 190 kDa multidrug resistance protein (MRP1).Although both MDR1 and MRP1 belong to the ATP-binding cassettesuperfamily of transport proteins, they are structurally very differentmolecules and share less than 15% amino acid homology. Despite thestructural divergence between the two proteins, by 1994 there were noknown consistent differences in the resistance patterns of MDR1 and MRP1cell lines. However, the association, or lack thereof, of MRP1 andresistance to particular oncolytics is known. See Cole, et. al.,Pharmacological Characterization of Multidrug Resistant MRP-transfectedHuman Tumor Cells, Cancer Research, 54:5902-5910, 1994. Doxorubicin,daunorubicin, epirubicin, vincristine, and etoposide are substrates ofMRP1, i.e., MRP1 can bind to these oncolytics and redistribute them awayfrom their site of action, the nucleus, and out of the cell. Id. andMarquardt, D., and Center, M. S., Cancer Research, 52:3157, 1992.

Doxorubicin, daunorubicin, and epirubicin are members of theanthracycline class of oncolytics. They are isolates of various strainsof Streptomyces and act by inhibiting nucleic acid synthesis. Theseagents are useful in treating neoplasms of the bone, ovaries, bladder,thyroid, and especially the breast. They are also useful in thetreatment of acute lymphoblastic and myeloblastic leukemia, Wilm'stumor, neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin'slymphomas, and bronchogenic carcinoma.

Vincristine, a member of the vinca alkaloid class of oncolytics, is anisolate of a common flowering herb, the periwinkle plant (Vinca roseaLinn). The mechanism of action of vincristine is still underinvestigation but has been related to the inhibition of microtubuleformation in the mitotic spindle. Vincristine is useful in the treatmentof acute leukemia, Hodgkin's disease, non-Hodgkin's malignant lymphomas,rhabdomyosarcoma, neuroblastoma, and Wilm's tumor.

Etoposide, a member of the epipodophyllotoxin class of oncolytics, is asemisynthetic derivative of podophyllotoxin. Etoposide acts as atopoisomerase inhibitor and is useful in the therapy of neoplasms of thetestis, and lung.

It is presently unknown what determines whether a cell line will acquireresistance via a MDR1 or MRP1 mechanism. Due to the tissue specificityof these transporters and/or in the case where one mechanismpredominates or is exclusive, it would be useful to have a selectiveinhibitor of that one over the other. Furthermore, when administering adrug or drugs that are substrates of either protein, it would beparticularly advantageous to co-administer an agent that is a selectiveinhibitor of that protein. It is, therefore, desirable to providecompounds which are selective inhibitors of MDR1 or MRP1.

The present invention relates to a compound of formula I:

where:

R is hydrogen, COR¹, SO₂R², or a moiety of the formula

R¹ is C₁-C₄ alkyl, aryl, substituted aryl, furanyl, indolyl,thiophenylmethyl, 5-methylisoxazolyl, NHR⁴, or CHR⁵OR⁶;

R² is 3,5-dimethylisoxazolyl or phenyl where the phenyl group isoptionally substituted once with nitro, C₁-C₄ alkyl, trifluoromethyl,C₁-C₄ alkoxy, C₂-C₄ alkanoyl, carbo(C₁-C₄ alkoxy), or amino(C₁-C₄alkyl);

R³ is phenyl where the phenyl group is optionally substituted once withtrifluoromethyl or N-acetylamino;

R⁴ is hydrogen, C₁-C₆ alkyl, or phenyl where the phenyl group isoptionally substituted once with nitro, C₁-C₄ alkyl, trifluoromethyl,C₁-C₄ alkoxy, C₂-C₄ alkanoyl, carbo(C₁-C₄ alkoxy), or amino(C₁-C₄alkyl);

R⁵ is hydrogen, C₁-C₄ alkyl, or phenyl; and

R⁶ is phenyl or acetyl; or a pharmaceutical salt or solvate thereof.

The present invention further relates to a method of inhibiting MRP1 ina mammal which comprises administering to a mammal in need thereof aneffective amount of a compound of formula I, or a pharmaceutical salt orsolvate thereof.

In another embodiment, the present invention relates to a method ofinhibiting a resistant neoplasm, or a neoplasm susceptible to resistancein a mammal which comprises administering to a mammal in need thereof aneffective amount of a compound of formula I, or a pharmaceutical salt orsolvate thereof, in combination with an effective amount of an oncolyticagent.

The present invention also relates to a pharmaceutical formulationcomprising a compound of formula I, or a pharmaceutical salt or solvatethereof, in combination with one or more oncolytics, pharmaceuticalcarriers, diluents, or excipients therefor.

The current invention concerns the discovery that a select group ofcompounds, those of formula I, are selective inhibitors of multidrugresistant protein (MRP1) and are thus useful in treating MRP1 conferredmultidrug resistance (MDR) in a resistant neoplasm and a neoplasmsusceptible to resistance.

The term “inhibit” as it relates to MRP1 and “inhibiting” MRP1′ refer toprohibiting, alleviating, ameliorating, halting, restraining, slowing orreversing the progression of, or reducing MRP1's ability to redistributean oncolytic away from the oncolytic's site of action, most often theneoplasm's nucleus, and out of the cell.

As used herein, the term “effective amount of a compound of formula I”refers to an amount of a compound of the present invention which iscapable of inhibiting MRP1. The term “effective amount of an oncolytic”refers to an amount of oncolytic capable of inhibiting a neoplasm,resistant or otherwise.

The term “inhibiting a resistant neoplasm, or a neoplasm susceptible toresistance” refers to prohibiting, halting, restraining, slowing orreversing the progression of, reducing the growth of, or killingresistant neoplasms and/or neoplasms susceptible to resistance.

The term “resistant neoplasm” refers to a neoplasm which is resistant tochemotherapy where that resistance is conferred in part, or in total, byMRP1. Such neoplasms include, but are not limited to, neoplasms of thebladder, bone, breast, lung(small-cell), testis, and thyroid and alsoincludes more particular types of cancer such as, but not limited to,acute lymphoblastic and myeloblastic leukemia, Wilm's tumor,neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin'slymphomas, and bronchogenic carcinoma.

A neoplasm which is “susceptible to resistance” is a neoplasm whereresistance is not inherent nor currently present but can be conferred byMRP1 after chemotherapy begins. Thus, the methods of this inventionencompass a prophylactic and therapeutic administration of a compound offormula I.

The term “chemotherapy” refers to the use of one or more oncolyticswhere at least one oncolytic is a substrate of MRP1. A “substrate ofMRP1” is an oncolytic that binds to MRP1 and is redistributed away fromthe oncolytics site of action, (the neoplasm's nucleus) and out of thecell, thus, rendering the therapy less effective.

The terms “treat” or “treating” bear their usual meaning which includespreventing, prohibiting, alleviating, ameliorating, halting,restraining, slowing or reversing the progression, or reducing theseverity of MRP1 derived drug resistance in a multidrug resistant tumor.

The compounds of formula I contain at least 2 chiral centers which arelocated at the 1 and 2 positions of the indane ring system. Theenantiomers with the absolute stereochemistry of R,R or S,S arecontemplated within the scope of the present invention. That is,compounds of formula I whose substituents at the 1 and 2 position of theindane ring that have a trans relationship to one another arecontemplated within the scope of the present invention.

In the general formulae of the present document, the general chemicalterms have their usual meanings. For example, the term “C₁-C₃ alkyl”refers to methyl, ethyl, propyl, isopropyl, and cyclopropyl. The term“C₁-C₄ alkyl” encompasses C₁-C₃ alkyl groups and also refers to butyl,cyclobutyl, s-butyl, and t-butyl. The term “C₁-C₆ alkyl” includes C₁-C₄alkyl groups and also refers to monovalent, straight, branched, orcyclic saturated hydrocarbon chains containing 5 or 6 carbon atomsincluding, but not limited to, cyclopentyl, pentyl, hexyl, cyclohexyl,and the like.

The term “C₂-C₄ alkanoyl” refers to a C₁-C₃ alkyl group attached througha carbonyl moiety.

The term “C₁-C₄ alkoxy” refers to a C₁-C₄ alkyl group attached throughan oxygen atom.

The term “halo” or “halide” refers to fluoro, chloro, bromo, or iodo.

The term “aryl” refers to phenyl, benzyl, and napthyl.

The term “substituted aryl” refers to a phenyl, benzyl, and napthylgroup, respectively, which is para substituted with a C₁-C₄ alkyl, C₁-C₄alkoxy, or halo group or is substituted independently three times with aC₁-C₄ alkoxy group.

The term “amino protecting group” as used in this specification refersto a substituent(s) of the amino group commonly employed to block orprotect the amino functionality while reacting other functional groupson the compound. Examples of such amino-protecting groups include theformyl group, the trityl group, the phthalimido group, the acetyl group,the trichloroacetyl group, the chloroacetyl, bromoacetyl, and iodoacetylgroups, urethane-type blocking groups such as benzyloxycarbonyl,9-fluorenylmethoxycarbonyl (“FMOC”), and the like; and like aminoprotecting groups. The species of amino protecting group employed is notcritical so long as the derivatized amino group is stable to thecondition of subsequent reaction(s) on other positions of the moleculeand can be removed at the appropriate point without disrupting theremainder of the molecule. Similar amino protecting groups used in thecephalosporin, penicillin, and peptide arts are also embraced by theabove terms. Further examples of groups referred to by the above termsare described by T. W. Greene, Protective Groups in Organic Synthesis,John Wiley and Sons, New York, N.Y., 1991, Chapter 7 hereafter referredto as Greene. A preferred amino protecting group is t-butyloxycarbonyl.

The term “carbonyl activating group” refers to a substituent of acarbonyl that renders that carbonyl prone to nucleophilic addition.Suitable activating groups are those which have a net electronwithdrawing effect on the carbonyl. Such groups include, but are notlimited to, alkoxy, aryloxy, nitrogen containing aromatic heterocycles,or amino groups such as oxybenzotriazole, imidazolyl, nitrophenoxy,pentachlorophenoxy, N-oxysuccinimide, N,N′-dicyclohexylisoure-O-yl,N-hydroxy-N-methoxyamino, and the like; acetates, formates, sulfonatessuch as methanesulfonate, ethanesulfonate, benzenesulfonate, orp-toluenylsulfonate, and the like; and halides especially chloride,bromide, or iodide.

In general, the term “pharmaceutical” when used as an adjective meanssubstantially non-toxic to living organisms. For example, the term“pharmaceutical salt” as used herein, refers to salts of the compoundsof formula I which are substantially non-toxic to living organisms. See,e.g., Berge, S. M, Bighley, L. D., and Monkhouse, D. C., pharmaceuticalsalts include those salts prepared by reaction of the compounds offormula I with an inorganic or organic acid. Such salts are known asacid addition salts. These pharmaceutical salts frequently have enhancedsolubility characteristics compared to the compound from which they arederived, and thus are often more amenable to formulation as liquids oremulsions.

Examples of pharmaceutical acid addition salts are the sulfate,pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,γ-hydroxybutyrate, glycollate, tartrate, methanesulfonate,ethanesulfonate, propanesulfonate, naphthalene-1-sulfonate,napththalene-2-sulfonate, mandelate, and the like of a compound offormula I.

The term “solvate” represents an aggregate that comprises one or moremolecules of the solute, such as a formula I compound, with one or moremolecules of solvent.

The term “suitable solvent” refers to a solvent which is inert to theongoing reaction and sufficiently solubilizes the reactants to effectthe desired reaction. Examples of suitable solvents include but are notlimited to, dichloromethane, chloroform, 1,2-dichloroethane, diethylether, acetonitrile, ethyl acetate, 1,3-dimethyl-2-imidazolidinone,tetrahydrofuran, dimethylformamide, toluene, chlorobenzene,dimethylsulfoxide, mixtures thereof, and the like.

The term “carbonyl activating reagent” refers to a reagent that convertsthe carbonyl of a carboxylic acid group to one that is more prone tonucleophilic addition and includes, but is not limited to, such reagentsas those found in The Peptides, Gross and Meienhofer, Eds., AcademicPress (1979), Ch. 2 and M. Bodanszky, Principles of Peptide Synthesis,2^(nd) Ed., Springer-Verlag Berlin Heidelberg, 1993, hereafter referredto as The Peptides and Peptide Synthesis respectively. Specifically,carbonyl activating reagents include nucleophilic sources of a halogensuch as, thionyl bromide, thionyl chloride, oxalyl chloride, and thelike; alcohols such as nitrophenol, pentachlorophenol, and the like;amines such as N-hydroxy-N-methoxyamine and the like; acid halides suchas acetic, formic, methanesulfonic, ethanesulfonic, benzenesulfonic, orp-tolenesulfonic acid halide, and the like; and compounds such as1,1′-carbonyldiimidazole, benzotriazole, imidazole,N-hydroxysuccinimide, dicyclohexylcarbodiimide, and the like.

The term “suitable thermodynamic base” refers to a base which acts as aproton trap for any protons which may be produced as a byproduct of thedesired reaction or to a base which provides a reversible deprotonationof an acidic substrate and is reactive enough to effect the desiredreaction without significantly effecting any undesired reactions.Examples of thermodynamic bases include, but are not limited to,carbonates, bicarbonates, and hydroxides (e.g., lithium, sodium, orpotassium carbonate, bicarbonate, or hydroxide), tri-(C₁-C₄alkyl)amines, or aromatic nitrogen containing heterocycles (e.g.,pyridine).

While all of the compounds of the present invention are useful, certainof the compounds are particularly interesting and are preferred. Thefollowing listing sets out several groups of preferred compounds,formulations, and methods. It will be understood that each of thelistings may be combined with other listings to create additional groupsof preferred embodiments.

a) R is COR¹;

b) R is SO₂R²;

c) R¹ is phenyl;

d) R¹ is benzyl;

e) R¹ is 3,4,5-trimethoxyphenyl;

f) R¹ is 3,4,5-trimethoxybenzyl;

g) R² is phenyl;

h) R² is 4-methoxyphenyl;

i) R² is 3,5-dimethoxyisoxazole;

j) The compound is a pharmaceutical salt;

k) The compound is the hydrochloride salt;

l) The compounds of the Examples section;

m) The method where the mammal is a human;

n) The method where the oncolytic(s) is selected from: doxorubicin,daunorubicin, epirubicin, vincristine, and etoposide;

o) The method where the neoplasm is of the Wilm's type, bladder, bone,breast, lung(small-cell), testis, or thyroid or the neoplasm isassociated with acute lymphoblastic and myeloblastic leukemia,neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin'slymphomas, or bronchogenic carcinoma;

p) The formulation where the oncolytic(s) is selected from the group:doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide;

q) The compound is the R isomer;

r) The compound is the S isomer; and

s) The compound is a mixture of isomers.

The compounds of the present invention can be prepared by a variety ofprocedures, including solid phase or solution phase synthetictechniques. Solid phase techniques are illustrated below in Scheme 1.The particular order of steps required to produce the compounds offormula I is dependent upon the particular compound being synthesized,the starting compound, and the relative lability of the substitutedmoieties.

The reactions of Scheme 1 are all performed in the solid phase. That is,the molecule that is manipulated in the various conversions is bound toan insoluble polystyrene resin and the reagents used to modify or add tothe bound molecule are soluble. A general strategy for syntheses of thiskind, which is applicable to this case, is to employ large molarexcesses of unbound reagents, relative to the bound reagent, in order toinsure complete conversion of the bound molecule. When the conversion issubstantially complete, the soluble reagents can simply be filteredaway. The impure resin which will contain residues of the just finishedreaction can be cleaned simply by rinsing the impure resin with, forexample, the same solvent employed in the reaction. For example, if thereaction was performed in tetrahydrofuran, you can rinse the resin withpure tetrahydrofuran. These principles are applicable to all thereactions discussed in Scheme 1.

Compounds of formula I may be prepared as illustrated in Scheme 1 below,where PS is a polystyrene resin, R⁵ is a carbonyl activating group, andR, R¹, R², and R³ are as described supra.

Acids of formula II may be activated to form the activated carboxylicacids of formula III by methods well known in the chemical arts. See,e.g., The Peptides or Peptide Synthesis for general discussions ofsolution phase activation and Preparations sections below for solidphase activation. Specifically, the compounds of formula III aretypically prepared by exposing a carboxylated polystyrene resin to amolar excess of activating reagent in a suitable solvent. A convenientand preferred solvent for this purpose is a mixture of dichloromethaneand dimethylformamide. Oxalyl chloride is typically a preferred andconvenient activating reagent and a 3 molar excess of this activatingreagent is generally employed. Usually the reaction is performed at thereflux temperature of the mixture for about 24 hours.

The resin bound esters of formula V may be prepared from compounds offormula IV and resin bound activated acids of formula III by methodsvery well known in the chemical arts. For a general instruction on thesolution phase conversion of activated carboxylic acids to esters see,e.g., Larock, Comprehensive Organic Transformations, pgs. 978-979, VCHPublishers, New York, N.Y., 1989, hereafter referred to as Larock.Specifically, the ester of formula III in the presence of athermodynamic base, optionally in the presence of a thermodynamic base,and optionally in the presence of dimethylamino pyridine (DMAP), may beexposed to the compound of formula IV. Tetrahydrofuran is typically aconvenient and preferred solvent. DMAP is preferably employed in acatalytic fashion, typically in about a 50 molar percent relative to thebound material. The compound of formula IV, however, is employed in anexcess, usually in about a 1.4 to about a 2.5 molar excess. A preferredbase is pyridine and is usually employed in large molar excesses,typically on the order of about a 20 molar excess. Once all the reagentsare combined, the reaction is usually allowed to proceed at the refluxtemperature of the mixture for 8 to 12 days.

The amino protecting group contained in the compound of formula V maynow be removed to form the compounds of formula VI. Choices ofprotecting groups and reagents and methods available to install orremove them may be found in the Greene reference cited above. Preferredprotecting groups and methods for their removal may be found in thePreparations section below.

The resin bound free amine of formula VI in the presence of a suitablesolvent, optionally in the presence of a thermodynamic base, andoptionally in the presence of dimethylamino pyridine (DMAP), may now betreated with a compound of formula VII to provide a compound of formulaX where R is COR¹. Typically a preferred and convenient solvent isdichloromethane. When a base is employed, pyridine is typically apreferred base. Furthermore, when a base is employed, the base andcompound of formula VII are typically employed in large stoichiometricexcesses relative to the resin bound material. For example the base istypically employed in between a 15 and 20 molar excess while thecompound of formula IV is generally employed in between a 5 to 8 molarexcess. When a base is not employed, the compound of formula VII istypically employed in a relatively larger stoichiometric excess. Thereaction is usually performed at a temperature range of about 0° C. toabout the reflux temperature of the solvent for from 10 minutes to 18hours. Preferably, the reaction is performed at about 15° C. to about40° C. for from 12 to 24 hours and most preferred is at room temperaturefor 18 hours.

Under the same conditions as the previous paragraph, a compound offormula VI may alternatively be treated with a compound of formula VIIIor IX to afford, respectively, the compounds of formula X where R isSO₂R² or a moiety of the formula

In the next reaction of Scheme 1, the nitro group at the 2 position ofthe indane ring is reduced to an amino moiety resulting in the compoundsof formula XI. Methods of reducing a nitro group to an amine are wellknown. See, e.g., Larock at 412-415 or the Preparations and Examplessections below. Usually, the transformation is accomplished by exposingthe resin bound compound of formula X to a large molar excess ofreducing agent in a suitable solvent. Dimethylformamide is typically aconvenient and preferred solvent for this purpose. Tin(II) chloridedihydrate is usually the preferred reducing agent. The reaction isusually performed at a temperature range of about 0° C. to about thereflux temperature of the solvent for from 12 hours to 72 hours.Preferably, the reaction is performed at about 15° C. to about 40° C.for from 30 to 60 hours and most preferred is at room temperature for 48hours.

The next reaction of Scheme 2 is an acylation of the amino moiety formedin the previous reaction to form the compounds of formula XIII. Thisacylation has the same reaction and reagent profiles of the conversionof compounds of formula VI to compounds of formula X except that in thiscase, the only acylating agent used is the compound of formula XII,i.e., a 3-(2-chloro-6-fluorophenyl)-5-methylisoxaz-4-oyl activated acid.

Finally, the compound of formula XIII may be cyclized and freed from theresin to form the compound of formula XIV. This transformation may beaccomplished by exposing the resin bound compound of formula XIII to asuitable thermodynamic base in a suitable solvent. Typically a preferredand convenient solvent is tetrahydrofuran. Usually a convenient andpreferred thermodynamic base is sodium hydroxide added as a 2N solutionin methanol. The reaction is typically performed at about 15° C. toabout the reflux temperature of the mixture for from 30 minutes to about18 hours. Preferably, the reaction is performed at room temperature forabout 18 hours. The base is typically employed in a large molar excess,usually in about a 10 to about a 15 molar excess relative to thecompound of formula XIV. Preferably, about a 11 to about a 13 molarexcess is typically employed.

Any amino protecting groups found in the cyclized compounds of formula Imay optionally be removed as taught in Greene to provide the free amine.Preferred choices of protecting groups and methods for their removal maybe found in the Preparations and Examples sections which follow.

The pharmaceutical salts of the invention are typically formed byreacting a compound of formula I with an equimolar or excess amount ofacid. The reactants are generally combined in a mutual solvent such asdiethylether, tetrahydrofuran, methanol, ethanol, isopropanol, benzene,and the like. The salts normally precipitate out of solution withinabout one hour to about ten days and can be isolated by filtration orother conventional methods.

Acids commonly employed to form pharmaceutical acid addition salts areinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, phosphoric acid, and the like, and organic acidssuch as p-toluenesulfonic, methanesulfonic acid, ethanesulfonic acid,oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid,citric acid, tartaric acid, benzoic acid, acetic acid, and the like.Preferred pharmaceutical acid addition salts are those formed withmineral acids such as hydrochloric acid, hydrobromic acid, and sulfuricacid, and those formed with organic acids such as maleic acid, tartaricacid, and methanesulfonic acid.

It should be recognized that the particular counterion forming a part ofany salt of this invention is not of a critical nature, so long as thesalt as a whole is pharmacologically acceptable and as long as thecounterion does not contribute undesired qualities to the salt as awhole.

Compounds of formula II, IV, VII, VIII, IX, and XII are known in the artand, to the extent not commercially available, are readily synthesizedby standard procedures commonly employed in the art. For example, thecompounds of formula IV may be prepared as taught in the Preparationssection below.

The optimal time for performing the reactions of Scheme 1 can bedetermined by monitoring the progress of the reaction via conventionalchromatographic techniques. Furthermore, it is preferred to conduct thereactions of the invention under an inert atmosphere, such as, forexample, argon, or, particularly, nitrogen. Choice of solvent isgenerally not critical so long as the solvent employed is inert to theongoing reaction and sufficiently solubilizes the reactants to effectthe desired reaction. The intermediate compounds of this invention arepreferably purified before their use in subsequent reactions.Purification of the intermediates typically entails washing the resin asdiscussed above. The compounds of formula I may be purified by theircrystallizing out of the reaction solution during their formation. Thesecompounds can then be collected by filtration. Alternatively, thereaction solvent may be removed by extraction, evaporation, ordecantation. These final products of formula I may be further purified,if desired by common techniques such as recrystallization orchromatography over solid supports such as silica gel or alumina.

The following Preparations and Examples are provided to better elucidatethe practice of the present invention and should not be interpreted inany way as to limit the scope of same. Those skilled in the art willrecognize that various modifications may be made while not departingfrom the spirit and scope of the invention. All publications mentionedin the specification are indicative of the level of those skilled in theart to which this invention pertains. The terms and abbreviations usedin the instant Preparations and Examples have their normal meaningsunless otherwise designated. For example ° C., N, mmol, g, mL, M, HPLC,MS(IS), MS(FAB), and H NMR, refer to degrees Celsius, normal ornormality, millimole or millimoles, gram or grams, milliliter ormilliliters, molar or molarity, high performance liquid chromatography,ion spray mass spectrometry, fast atom bombardment mass spectrometry,and proton nuclear magnetic resonance spectrometry respectively.

Preparations Preparation 1 6-Nitro-1-Indanone

To a solution of 1-indanone (25.0 g, 189 mmol) in concentrated sulfuricacid (84 mL) at 0° C. was added a solution of potassium nitrate (8.33 g,82.4 mmol) in sulfuric acid (40 mL) at a rate sufficient to maintain aninternal temperature below 15° C. After the addition was complete, thereaction was allowed to stir at 0° C. for 1 hour. The reaction mixturewas then poured into crushed ice and stirred vigorously for 30 minutes.The suspension was then filtered, air dried, and purified by liquidchromatography (50 ethyl acetate/toluene) to provide 18.90 g of thetitle compound. (56%).

Preparation 2 6-Nitro-1-Indanol

A solution of 6-nitro-1-indanone (18.9 g, 107 mmol) in methanol (300 mL)was cooled to 0° C. and sodium borohydride (4.04 g, 107 mmol) was addedin several small portions. The reaction was then stirred overnight at25° C. The solution was quenched at 0° C. with methanolic hydrochloricacid (200 mL), concentrated under reduced pressure, redissolved indichloromethane, washed with water, and the organic layer reconcentratedto provide the crude alcohol as a brown solid which was used withoutfurther purification in Preparation 3.

Preparation 3 6-Nitro-1-Indene

To a solution of 6-nitro-1-indanol in toluene (300 mL) was added acatalytic amount of p-toluenesulfonic acid and the reaction was refluxedfor 1 hour using a Dean Stark trap to remove the water. The organiclayer was washed with saturated aqueous sodium bicarbonate (3×200 mL),dried over magnesium sulfate, filtered, and the filtrate solvent removedunder vacuum. The crude residue was crystallized from methanol to afford13.41 g of the title compound. (78% over two steps).

Preparation 4 6-Nitro-1,2-Epoxyindane

To a solution of 6-nitro-1-indene (10.5 g, 65.3 mmol) in dichloromethane(350 mL) at 0° C. was added meta chloroperbenzoic acid (29.0 g, 92.4mmol) in small amounts over the course of 1 hour. After stirringovernight at 25° C., the mixture was washed with saturated aqueoussodium sulfite (2×200 mL) and saturated aqueous sodium bicarbonate(2×200 mL), filtered through a cotton plug, and concentrated undervacuum to give the title compound which was used in Preparation 5without further purification.

Preparation 5 Trans-6-Nitro-1-Amino-2-Hydroxyindane

A suspension of 6-nitro-1,2-epoxyindane in concentrated ammoniumhydroxide (250 mL) was heated overnight in an oil bath at 45° C. Thenext day water was added and the basic aqueous layer was saturated withsodium chloride. The cloudy reaction mixture was extracted withtetrahydrofuran until no more product could be seen in the aqueous layerby TLC. The organic layers were combined, dried over magnesium sulfate,filtered, concentrated, and recrystallized from ethyl acetate to give11.54 g of the title compound. (91% over two steps).

Preparation 6 Trans-N-t-Butyloxycarbonyl-6-Nitro-1-Amino-2-Hydroxyindane

To a solution of trans-6-nitro-1-amino-2-hydroxyindane (8.34 g, 42.9mmol) in tetrahydrofuran (200 mL) was added a solution ofdi-t-butyldicarbonate (11.3 g, 51.5 mol) in tetrahydrofuran (50 mL).After stirring 1 hour at 25° C., the solvent was removed under reducedpressure and the resulting solid was recrystallized from ethyl acetateto afford 11.37 g of the title compound. (90%).

Preparation 7 Solid Phase Synthesis of Trans O-Resin Bound1-Amino-2-Hydroxy-6-Nitroindane

Step 1: Activation of the Resin/Binding to the Resin—Trans O-Resin BoundN-t-Butyloxycarbonyl-1-Amino-2-Hydroxy-6-Nitroindane

Under an nitrogen atmosphere, a 3 L three-necked round bottomed flaskequipped with an overhead stirrer and addition funnel was charged withcarboxylated polystyrene resin (70 g, 2.8 mmol CO₂H/g resin), anhydrousdichloromethane (1 L), and anhydrous dimethylformamide (10 mL). Next,oxalyl chloride (50.8 mL, 582 mmol) was added via a slow dropwiseaddition from an addition funnel. After refluxing overnight undernitrogen, the solvent was removed under vacuum using a gas dispersiontube. The resin was subsequently washed with anhydrous dichloromethane(3×500 mL). Once the last wash was complete, the resin was dried undervacuum for 2 to 3 hours. At this time, the polymer was resuspended indry tetrahydrofuran (1 L) followed by the addition of dry pyridine (314mL, 3.88 mol), DMAP (12 g, 97 mmol), and trans O-resin bound1-amino-2-hydroxy-6-nitroindane (85.6 g, 291 mmol). The mixture wasrefluxed for 10 days under an inert atmosphere. The solvent was removedby vacuum filtration and the resin was washed with tetrahydrofuran(3×300 mL), dichloromethane (3×300 mL), and dried overnight in a vacuumoven to provide 122.18 g of the title compound as a tan resin.

Step 2: Deprotection—Trans O-Resin Bound 1-Amino-2-Hydroxy-6-Nitroindane

Into a round bottomed flask equipped with a stir bar was placed thetrans O-resin bound N-t-butyloxycarbonyl-1-amino-2-hydroxy-6-nitroindane(28 mg, 0.028 mmol), 500 μl dichloromethane, and trifluoroacetic acid(109 μl, 0.141 mmol). The reaction mixture was stirred at 25° C.overnight. The resin was then collected by filtration, resuspended in10% triethylamine/dichloromethane, stirred for 15 minutes, filteredagain, and finally washed with dichloromethane to afford the titlecompound.

General Procedure for Preparations 8-36 Trans O-Resin boundN-“R”-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)-1-Amino-2-Hydroxyindane

Step 1: Installation of the “R” Moiety—Trans O-Resin BoundN-“R”-1-Amino-2-Hydroxy-6-Nitroindane

Into a 10 mL round bottom flask was placed trans O-resin bound1-amino-2-hydroxy-6-nitroindane (0.0283 mmol) followed by 500 μL of asolution of pyridine (36.6 μL, 0.452 mmol) and DMAP (0.518 mg, 0.00424mmol) in dichloromethane. Next, a 1M solution of a commerciallyavailable compound of formula VI, VII, or VIII in dichloromethane (184μL, 0.184 mmol) was added and the resulting mixture was stirredovernight at 25° C. At this time, the solvent was removed by vacuumfiltration and the resin was washed with 50 mL each of dichloromethane,dimethylformamide, methanol, dimethylformamide, methanol, anddichloromethane to give the title compound (compounds of formula X).

Step 2: Reduction—Trans O-Resin Bound6-Amino-1-((N-“R”)-Amino-2-Hydroxyindane

To a solution of trans O-resin boundN-“R”-1-amino-2-hydroxy-6-nitroindane (0.0283 mmol) in dimethylformamide(0.625 mL) was added tin(II)chloride dihydrate (102 mg, 0.452 mmol).Upon stirring at 25° C. for 48 hours, the resin was isolated byfiltration and washed with 50 mL each of dichloromethane,dimethylformamide, methanol, dimethylformamide, methanol, anddichloromethane to give the title compound (compounds of formula XI).

Step 3: Acylation—Trans O-Resin boundN-“R”-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)-1-Amino-2-Hydroxyindane

Into a 10 mL round bottomed flask was placed trans O-Resin Bound6-amino-1-((N-“R”)-amino)-2-hydroxyindane (0.0283 mmol) followed by 500μL of a solution of pyridine (36.6 μL, 0.452 mmol) and DMAP (0.518 mg,0.00424 mmol) in dichloromethane. Next a 1M solution of3-(2-chloro-6-fluorophenyl)5-methylisoxaz-4-oyl chloride indichloromethane (184 μL, 0.184 mmol) was added and the resulting mixturewas stirred overnight at 25° C. At this time, the solvent was removed byvacuum filtration and the resin was washed with 50 mL each ofdichloromethane, dimethylformamide, methanol, dimethylformamide,methanol, and dichloromethane to give the diacylated product (compoundof formula XIII).

Preparations 8-36 correspond to the precursors for Examples 1-29 below.

Preparation 37Trans-6-Amino-1-((N-t-Butyloxycarbonyl)-Amino)-2-Hydroxyindane

Palladium on carbon (10%, 500 mg) was wetted with methanol thentrans-N-t-butyloxycarbonyl-6-nitro-1-amino-2-hydroxyindane (2.63 g, 8.94mmol) dissolved in 100 mL of methanol was added to it. At 50 psi, anatmosphere of hydrogen was created and maintained in a Parr shaker forabout 18 hours. The catalyst was filtered off through talc. and washedwith methanol. The methanol was removed in vacuo leaving 2.23 g of thetitle compound. (94%). MS(FD) m/z 264 (M+). IR(CHCl₃) 3444, 3009, 2983,1692, 1625 cm⁻¹.

Preparation 381-(Trans-1-((N-t-Butyloxycarbonyl)-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-one

Trans-1-((N-t-butyloxycarbonyl)-amino)-6-amino-2-hydroxyindane (4 g,7.97 mmol) was dissolved in 10 mL of 2N sodium hydroxide in methanol andstirred under nitrogen at room temperature for about 18 hours. Thereaction was diluted with ethyl acetate, washed with 1N aqueoushydrochloric acid and brine, dried over sodium sulfate, filtered, andconcentrated. The residue was crystallized from dichloromethane to give4.07 g of the title compound. (100%). EA calculated for C₂₅H₂₄ClN₃O₅: C,62.31; H, 5.02; N, 8.72. Found: C, 62.11; H, 5.08, N, 8.65. MS(FD) m/z481 (M−H).

Preparation 391-(Trans-1-Amino-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneTrifluoroacetate

1-(Trans-1-((N-t-butyloxycarbonyl)-amino)-2-hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-one(759 mg, 1.57 mmol) was dissolved in 25 mL of dry dichloromethane andstirred under nitrogen at room temperature. Trifluoroacetic acid (10 mL)was added and the reaction was stirred for 1 hour. The solvents werethen removed in vacuo, and the residue was treated withdichloromethane/hexanes to precipitate 850 mg of the title compound.(100%). MS(FD) m/z 381 (M+). IR(KBr) 3058, 2998, 1792, 1657, 1629, 1598cm⁻¹.

EXAMPLES General Procedure for Examples 1-29

To a flask containing trans O-resin boundN-“R”-6-(isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-onyl)-1-amino-2-hydroxyindane(0.0283 mmol) was added a 1M solution of sodium hydroxide in methanol(375 μL, 0.375 mmol) and tetrahydrofuran (400 μL). After stirring at 25°C., the reaction was neutralized with 4M hydrochloric acid in methanol(100 μL, 0.400 mmol) The resin was filtered and the filtrate wasconcentrated under reduced pressure to provide the compound of Examples1-29 which gave satisfactory ¹H NMR and MS(IS) analysis.

Example 11-(Trans-1-((N-Acetyl)-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 21-(Trans-1-((N-Benzoyl)-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 31-(Trans-1-((N-[4-Methylbenzoyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 41-(Trans-1-((N-[4-Methoxybenzoyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 51-(Trans-1-((N-[4-Fluorobenzoyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 61-(Trans-1-((N-[4-Chlorobenzoyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 71-(Trans-1-((N-[α-Phenylacetyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 81-(Trans-1-((N-[α-(4-Chlorophenyl)acetyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 91-(Trans-1-((N-[α-Phenoxyacetyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 101-(Trans-1-((N-[2-Phenoxybutanoyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 111-(Trans-1-((N-[α-Phenyl-α-Acetoxyacetyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 121-(Trans-1-((N-Napthoyl)-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 13N-n-Butyl-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylUrea Example 14N-Cyclohexyl-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylUrea Example 15N-Phenyl-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylUrea Example 16N-(4-Isopropylphenyl)-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylUrea Example 17N-(3-Trifluoromethylphenyl)-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylUrea Example 18N-(4-Methoxyphenyl)-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylUrea Example 19N-(3-Acetylphenyl)-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylUrea Example 20N-(3-(Aminomethylphenyl)-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylUrea Example 21N-(3-Carboethoxyphenyl)-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylUrea Example 221-(Trans-1-((N-[3-Nitrophenylsulfonyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 231-(Trans-1-((N-[(5-Methylisoxazol-4-yl)sulfonyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 24N-(3-Trifluorophenyl)-N′-(Trans-2-Hydroxy-6-(isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylIsothiourea Example 25N-(N-Acetyl-3-Aminophenyl)-N′-(Trans-2-Hydroxy-6-(Isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-onyl)indanylIsothiourea Example 261-(Trans-1-((N-[Furan-2-oyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 271-(Trans-1-((N-[1H-Indol-3-oyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 281-(Trans-1-((N-[α-(Thiophen-2-yl)acetyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-oneExample 291-(Trans-1-((N-[5-Methylisoxazol-3-oyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-one

MS(FAB) m/z 491 (M+1). ¹H NMR (CDCl₃): δ2.4 (1.8H, s), 2.45 (1.2H, s),2.85 (1.8H, s), 2.9 (1.2H, s), 3.0-3.1 (1H, m), 3.3-3.5 (1H, m), 4.25(1H, bs), 4.3-4.4 (0.6H, m), 4.6-4.7 (0.4H, m), 5.3-5.4 (0.4H, m),5.45.5 (0.6H, m), 6.35 (0.6H, s), 6.42 (0.4H, s), 6.54 (0.4H, d), 6.6(0.6H, d), 7.15-7.55 (5H, m)

Example 301-(Trans-1-((N-[3,4,5-Trimethoxybenzoyl])-Amino)-2-Hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-Dihydro-3-methyl-6-chloroquinolin-2-one

1-(Trans-1-amino-2-hydroxyindan-6-yl)isoxazolo[3,4-c]-1,2-dihydro-3-methyl-6-chloroquinolin-2-onetrifluoroacetate (305 mg, 0.615 mmol) was dissolved in 10 mL of drydimethylformamide and stirred at room temperature under nitrogen.3,4,5-Trimethoxybenzoyl chloride (142 mg, 0.615 mmol) was then addedfollowed by triethylamine (187 mg, 1.85 mmol). The reaction was stirredfor about 18 hours and worked up as follows: diluted reaction with ethylacetate and 1N aqueous hydrochloric acid, washed the organic layer twicewith 1N aqueous hydrochloric acid, washed the organic layer thrice eachwith sodium bicarbonate and brine, and dried the organic layer oversodium sulfate. The ethyl acetate was removed and the white solid wastriturated with dichloromethane/hexanes to give 275 mg of the titlecompound. (78%). MS(IS) 576 (M+). EA calculated for C₃₀H₂₆ClN₃O₇: C,62.56; H, 4.55; N, 7.30. Found: C, 62.41; H, 4.40; N, 7.19.

The compounds of the invention are inhibitors of MRP1. Thus, thecompounds of the invention may be used to inhibit any neoplasm havingintrinsic and/or acquired resistance, conferred in part or in total byMRP1, to an oncolytic or oncolytics. In other words, treatment of such aneoplasm with an effective amount of a compound of this invention willcause the neoplasm to be more sensitive to chemotherapy that wasrendered less efficacious by MRP1.

Vincristine, epirubicin, daunorubicin, doxorubicin, and etoposide areoncolytics that are substrates of MRP1. See Cole, et. al.,Pharmacological Characterization of Multidrug Resistant MRP-transfectedHuman Tumor Cells, Cancer Research, 54:5902-5910, 1994. Since MRP1 isubiquitous in mammals, particularly humans, Nooter, K, et. al.,Expression of the Multidrug Resistance-Associated Protein (MRP) Gene inHuman Cancers, Clin. Can. Res., 1:1301-1310, (1995), chemotherapy whosegoal is to inhibit a neoplasm employing any of those agents has thepotential to be rendered less efficacious by MRP1. Thus, neoplasms ofthe bladder, bone, breast, lung(small-cell), testis, and thyroid andmore specific types of cancer such as acute lymphoblastic andmyeloblastic leukemia, Wilm's tumor, neuroblastoma, soft tissue sarcoma,Hodgkin's and non-Hodgkin's lymphomas, and bronchogenic carcinoma may beinhibited with a combination of one or more of the above oncolytics anda compound of this invention.

The biological activity of the compounds of the present invention wasevaluated employing an initial screening assay which rapidly andaccurately measured the activity of the tested compound in inhibitingMRP1 or MDR1. Assays useful for evaluating this reversing capability arewell known in the art. See, e.g., T. McGrath, et al., BiochemicalPharmacology, 38:3611, 1989; D. Marquardt and M. S. Center, CancerResearch, 52:3157, 1992; D. Marquardt, et al., Cancer Research, 50:1426,1990; and Cole, et. al., Cancer Research, 54:5902-5910, 1994.

Assay for Reversal of MRP1-Mediated Doxorubicin Resistance andMDR1-Mediated Vincristine Resistance: HL60/ADR and HL60/VCR arecontinuous cell lines, which were selected for doxorubicin andvincristine resistance, respectively, by culturing HL60, a human acutemyeloblastic leukemia cell line, in increasing concentrations ofdoxorubicin or vincristine until a highly resistant variant wasattained.

HL60/ADR and HL60/VCR cells were grown in RPMI 1640 (Gibco) containing10% fetal bovine serum (FBS) and 250 μg/mL GENTAMICIN™ (Sigma) cellswere harvested; washed twice with assay medium (same as culture media);counted; and diluted to 2×10⁵ cells/mL in assay medium. Fiftymicroliters of cells were aliquoted into wells of a 96 well tissueculture plate. One column of each 96 well plate served as a negativecontrol and received assay medium containing no cells.

Test compounds and reference compounds were dissolved in dimethylsulfoxide (DMSO) at a concentration of 5 mM. Samples were diluted to 20μM in assay medium and 25 μl of each test compound was added to 6 wells.Assay standards were run in quadruplicate. Twenty-five microliters of0.4% DMSO was added to four wells as a solvent control. Assay media wasadded to all wells to achieve a final volume of 100 μl per well.

The plates were incubated at 37° C. for 72 hours in a humidifiedincubator with a 5% carbon dioxide atmosphere. Cell viability andvitality was measured by oxidation of a tetrazolium salt suing standardconditions. The plates were incubated for 3 hours at 37° C. Absorbancewas determined at 490 nm using a microtitre plate reader.

The ability of a test compound to reverse the resistance of HL60/ADR andHL60/VCR cells to doxorubicin was determined by comparison of theabsorbance of the wells containing a test compound in addition to theoncolytic (doxorubicin) with the absorbance of wells containing theoncolytic without a test compound. Controls were used to eliminatebackground and to ensure the results were not artifactual. The resultsof the assay are expressed as percent inhibition of cell growth. Theoncolytic alone at the tested concentration does not usually inhibit thegrowth of HL60/ADR or HL60/VCR cells.

Representative compounds of formula I demonstrated a significant effectin reversing the MRP1 multiple drug resistance. Many of the compoundsshowed very significant enhancement of activity in combination with theoncolytic agent as opposed to the oncolytic agent alone. In addition, alarge majority of the compounds tested displayed a significant degree ofselective inhibition of the HL60/ADR cell line over the HL60/VCR cellline.

When administering an oncolytic in practicing the methods of thisinvention, the amount of oncolytic employed will be variable. It shouldbe understood that the amount of the oncolytic actually administeredwill be determined by a physician, in the light of the relevantcircumstances, including the condition to be treated, the chosen routeof administration, the actual oncolytic administered, the age, weight,and response of the individual patient (mammal), and the severity of thepatient's symptoms. Of course, the amount of oncolytic administeredshould be decided and closely monitored by that patient's physician.After deciding on the oncolytic or oncolytics to employ, The Physician'sDesk Reference, published by Medical Economics Company at Montvale, N.J.07645-1742, is a helpful resource to the physician in deciding onamounts of the oncolytic to administer and is updated annually.

Preferred formulations, and the methods of this invention employingthose formulations, are those which do not contain an oncolytic. Thus,it is preferred to administer the compounds of this invention separatelyfrom the oncolytic. The oncolytics mentioned in this specification arecommercially available and may be purchased in pre-formulated formssuitable for the methods of this invention.

The compounds of formula I alone, or optionally in combination with anoncolytic, are usually administered in the form of pharmaceuticalformulations. These formulations can be administered by a variety ofroutes including oral, rectal, transdermal, subcutaneous, intravenous,intramuscular, and intranasal. Such formulations are prepared in amanner well known in the pharmaceutical art and comprise at least oneactive compound of formula I.

The present invention also includes methods employing pharmaceuticalformulations which contain, as the active ingredient, the compounds offormula I, and optionally an oncolytic, associated with pharmaceuticalcarriers. In making the formulations of the present invention the activeingredient(s) is usually mixed with an excipient, diluted by anexcipient, or enclosed within such a carrier which can be in the form ofa capsule, sachet, paper or other container. When the excipient servesas a diluent, it can be a solid, semi-solid, or liquid material, whichacts as a vehicle, carrier or medium for the active ingredient. Thus,the formulations can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,syrups, aerosols (as a solid or in a liquid medium), ointmentscontaining for example up to 10% by weight of the active compound, softand hard gelatin capsules, suppositories, sterile injectable solutions,and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound(s) to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound(s) is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound(s) is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g., about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxybenzoates; sweetening agents; and flavoring agents. Theformulations of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The formulations are preferably formulated in a unit dosage form, eachdosage containing from about 5 to about 100 mg, more usually about 10 toabout 30 mg, of each active ingredient. The term “unit dosage form”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

The compounds of formula I are effective over a wide dosage range. Forexample, dosages per day normally fall within the range of about 0.5 toabout 30 mg/kg of body weight. In the treatment of adult humans, therange of about 1 to about 15 mg/kg/day, in single or divided dose, isespecially preferred. However, it will be understood that the amount ofthe compound actually administered will be determined by a physician, inthe light of the relevant circumstances, including the condition to betreated, the chosen route of administration, the actual compoundadministered, the age, weight, and response of the individual patient,and the severity of the patient's symptoms, and therefore the abovedosage ranges are not intended to limit the scope of the invention inany way. In some instances dosage levels below the lower limit of theaforesaid range may be more than adequate, while in other cases stilllarger doses may be employed without causing any harmful side effect,provided that such larger doses are first divided into several smallerdoses for administration throughout the day.

For preparing solid formulations such as tablets the principal activeingredient(s) is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient(s) is dispersed evenly throughout the formulation so that theformulation may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The novel formulations which are liquid forms may be incorporated foradministration orally or by injection and include aqueous solutions,suitably flavored syrups, aqueous or oil suspensions, and flavoredemulsions with edible oils such as cottonseed oil, sesame oil, coconutoil, or peanut oil, as well as elixirs and similar pharmaceuticalvehicles.

Formulations for inhalation or insufflation include solutions andsuspensions in pharmaceutical, aqueous or organic solvents, or mixturesthereof, and powders. The liquid or solid formulations may containsuitable pharmaceutical excipients as described supra. Preferably theformulations are administered by the oral or nasal respiratory route forlocal or systemic effect. Compositions in preferably pharmaceuticalsolvents may be nebulized by use of inert gases. Nebulized solutions maybe breathed directly from the nebulizing device or the nebulizing devicemay be attached to a face mask, tent, or intermittent positive pressurebreathing machine. Solution, suspension, or powder formulations may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way. “ActiveIngredient(s)” means a compound according to formula I or apharmaceutical salt or solvate thereof optionally with one or moreoncolytics.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient (s)  30.0 Starch305.0 Magnesium stearate  5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mq/tablet) Active Ingredient (s)  25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide  10.0 Stearic acid  5.0

The components are blended and compressed to form tablets, each weighing240 mg.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Active Ingredient (s)  5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows:

Quantity Ingredient (mg/tablet) Active Ingredient (s) 30.0 mg Starch45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone (as 10%solution in water)  4.0 mg Sodium carboxymethyl starch  4.5 mg Magnesiumstearate  0.5 mg Talc  1.0 mg Total  120 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50-60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

Formulation Example 5

Capsules, each containing 40 mg of medicament are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient (s)  40.0 mg Starch109.0 mg Magnesium stearate  1.0 mg Total 150.0 mg

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 150 mg quantities.

Formulation Example 6

Suppositories, each containing 25 mg of active ingredient are made asfollows:

Ingredient Amount Active Ingredient (s)   25 mg Saturated fatty acidglycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 7

Suspensions, each containing 50 mg of medicament per 5.0 mL dose aremade as follows:

Ingredient Amount Active Ingredient (s) 50.0 mg Xanthan gum 4.0 mgSodium carboxymethyl cellulose (11%) 50.0 mg Microcrystalline cellulose(89%) Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v.Purified water to 5.0 mL

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

Formulation Example 8

Capsules, each containing 15 mg of medicament, are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient (s)  15.0 mg Starch407.0 mg Magnesium stearate  3.0 mg Total 425.0 mg

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 425 mg quantities.

Formulation Example 9

An intravenous formulation may be prepared as follows:

Ingredient Quantity Active Ingredient (s) 250.0 mg Isotonic saline  1000mL

Formulation Example 10

A topical formulation may be prepared as follows:

Ingredient Quantity Active Ingredient (s) 1-10 g Emulsifying Wax 30 gLiquid Paraffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

Formulation Example 11

Sublingual or buccal tablets, each containing 10 mg of activeingredient, may be prepared as follows:

Quantity Ingredient Per Tablet Active Ingredient (s)  10.0 mg Glycerol210.5 mg Water 143.0 mg Sodium Citrate  4.5 mg Polyvinyl Alcohol  26.5mg Polyvinylpyrrolidone  15.5 mg Total 410.0 mg

The glycerol, water, sodium citrate, polyvinyl alcohol, andpolyvinylpyrrolidone are admixed together by continuous stirring andmaintaining the temperature at about 90° C. When the polymers have goneinto solution, the solution is cooled to about 50-55° C. and the activeingredient is slowly admixed. The homogenous mixture is poured intoforms made of an inert material to produce a drug-containing diffusionmatrix having a thickness of about 2-4 mm. This diffusion matrix is thencut to form individual tablets having the appropriate size.

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

Frequently, it will be desirable or necessary to introduce thepharmaceutical formulation to the brain, either directly or indirectly.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system, used for the transport ofbiological factors to specific anatomical regions of the body, isdescribed in U.S. Pat. No. 5,011,472, issued Apr. 30, 1991, which isherein incorporated by reference.

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs or prodrugs.Latentiation is generally achieved through blocking of the hydroxy,carbonyl, sulfate, and primary amine groups present on the drug torender the drug more lipid soluble and amenable to transportation acrossthe blood-brain barrier. Alternatively, the delivery of hydrophilicdrugs may be enhanced by intra-arterial infusion of hypertonic solutionswhich can transiently open the blood-brain barrier.

We claim:
 1. A compound of formula I:

where: R is hydrogen, COR¹, SO₂R², or a moiety of the formula

R¹ is C₁-C₄ alkyl, aryl, substituted aryl, furanyl, indolyl,thiophenylmethyl, 5-methylisoxazolyl, NHR⁴, or CHR⁵OR⁶; R² is3,5-dimethylisoxazolyl or phenyl where the phenyl group is optionallysubstituted once with nitro, C₁-C₄ alkyl, trifluoromethyl, C₁-C₄ alkoxy,C₂-C₄ alkanoyl, carbo(C₁-C₄ alkoxy), or amino(C₁-C₄ alkyl); R³ is phenylwhere the phenyl group is optionally substituted once withtrifluoromethyl or N-acetylamino; R⁴ is hydrogen, C₁-C₆ alkyl, or phenylwhere the phenyl group is optionally substituted once with nitro, C₁-C₄alkyl, trifluoromethyl, C₁-C₄ alkoxy, C₂-C₄ alkanoyl, carbo(C₁-C₄alkoxy), or amino(C₁-C₄ alkyl); R⁵ is hydrogen, C₁-C₄ alkyl, or phenyl;and R⁶ is phenyl or acetyl; or a pharmaceutical salt or solvate thereof.2. The compound according to claim 1 where R is COR¹ and R¹ is3,4,5-trimethoxyphenyl.
 3. A method of inhibiting MRP1 in a mammal whichcomprises administering to a mammal in need thereof an effective amountof a compound of formula I:

where: R is hydrogen, COR¹, SO₂R², or a moiety of the formula

R¹ is C₁-C₄ alkyl, aryl, substituted aryl, furanyl, indolyl,thiophenylmethyl, 5-methylisoxazolyl, NHR⁴, or CHR⁵OR⁶; R² is3,5-dimethylisoxazolyl or phenyl where the phenyl group is optionallysubstituted once with nitro, C₁-C₄ alkyl, trifluoromethyl, C₁-C₄ alkoxy,C₂-C₄ alkanoyl, carbo(C₁-C₄ alkoxy), or amino(C₁-C₄ alkyl); R³ is phenylwhere the phenyl group is optionally substituted once withtrifluoromethyl or N-acetylamino; R⁴ is hydrogen, C₁-C₆ alkyl, or phenylwhere the phenyl group is optionally substituted once with nitro, C₁-C₄alkyl, trifluoromethyl, C₁-C₄ alkoxy, C₂-C₄ alkanoyl, carbo(C₁-C₄alkoxy), or amino(C₁-C₄ alkyl); R⁵ is hydrogen, C₁-C₄ alkyl, or phenyl;and R⁶ is phenyl or acetyl; or a pharmaceutical salt or solvate thereof.4. The method according to claim 2 where the mammal is a human.
 5. Themethod according to claim 4 where the compound of formula I is acompound where R is COR¹ and R¹ is 3,4,5-trimethoxyphenyl.
 6. A methodof inhibiting a resistant neoplasm, or a neoplasm susceptible toresistance, in a mammal which comprises administering to a mammal inneed thereof an effective amount of a compound of formula I:

where: R is hydrogen, COR¹, SO₂R², or a moiety of the formula

R¹ is C₁-C₄ alkyl, aryl, substituted aryl, furanyl, indolyl,thiophenylmethyl, 5-methylisoxazolyl, NHR⁴, or CHR⁵OR⁶; R² is3,5-dimethylisoxazolyl or phenyl where the phenyl group is optionallysubstituted once with nitro, C₁-C₄ alkyl, trifluoromethyl, C₁-C₄ alkoxy,C₂-C₄ alkanoyl, carbo(C₁-C₄ alkoxy), or amino(C₁-C₄ alkyl); R³ is phenylwhere the phenyl group is optionally substituted once withtrifluoromethyl or N-acetylamino; R⁴ is hydrogen, C₁-C₆ alkyl, or phenylwhere the phenyl group is optionally substituted once with nitro, C₁-C₄alkyl, trifluoromethyl, C₁-C₄ alkoxy, C₂-C₄ alkanoyl, carbo(C₁-C₄alkoxy), or amino(C₁-C₄ alkyl); R⁵ is hydrogen, C₁-C₄ alkyl, or phenyl;and R⁶ is phenyl or acetyl; or a pharmaceutical salt or solvate thereof;in combination with an effective amount of one or more oncolytic agents.7. The method according to claim 6 where the mammal is a human.
 8. Themethod according to claim 7 where the oncolytic(s) is selected from thegroup: doxorubicin, daunorubicin, epirubicin, vincristine, andetoposide.
 9. The method according to claim 7 where the neoplasm is aneoplasm of the Wilm's type, bladder, bone, breast, lung(small-cell),testis, or thyroid or the neoplasm is associated with acutelymphoblastic and myeloblastic leukemia, neuroblastoma, soft tissuesarcoma, Hodgkin's and non-Hodgkin's lymphomas, or bronchogeniccarcinoma.
 10. The method according to any of claims 7-9 where thecompound of formula I is a compound where R is COR¹ and R¹ is3,4,5-trimethoxyphenyl.
 11. A pharmaceutical formulation comprising acompound of formula I:

where: R is hydrogen, COR¹, SO₂R², or a moiety of the formula

R¹ is C₁-C₄ alkyl, aryl, substituted aryl, furanyl, indolyl,thiophenylmethyl, 5-methylisoxazolyl, NHR⁴, or CHR⁵OR⁶; R² is3,5-dimethylisoxazolyl or phenyl where the phenyl group is optionallysubstituted once with nitro, C₁-C₄ alkyl, trifluoromethyl, C₁-C₄ alkoxy,C₂-C₄ alkanoyl, carbo(C₁-C₄ alkoxy), or amino(C₁-C₄ alkyl); R³ is phenylwhere the phenyl group is optionally substituted once withtrifluoromethyl or N-acetylamino; R⁴ is hydrogen, C₁-C₆ alkyl, or phenylwhere the phenyl group is optionally substituted once with nitro, C₁-C₄alkyl, trifluoromethyl, C₁-C₄ alkoxy, C₂-C₄ alkanoyl, carbo(C₁-C₄alkoxy), or amino(C₁-C₄ alkyl); R⁵ is hydrogen, C₁-C₄ alkyl, or phenyl;and R⁶ is phenyl or acetyl; or a pharmaceutical salt or solvate thereof;in combination with one or more pharmaceutical carriers, diluents, orexcipients therefor.
 12. The formulation according to claim 11 where thecompound of formula I is a compound where R is COR¹ and R¹ is3,4,5-trimethoxyphenyl.
 13. A pharmaceutical formulation comprising: (a)a compound of formula I:

 where: R is hydrogen, COR¹, SO₂R², or a moiety of the formula

R¹ is C₁-C₄ alkyl, aryl, substituted aryl, furanyl, indolyl,thiophenylmethyl, 5-methylisoxazolyl, NHR⁴, or CHR⁵OR⁶; R² is3,5-dimethylisoxazolyl or phenyl where the phenyl group is optionallysubstituted once with nitro, C₁-C₄ alkyl, trifluoromethyl, C₁-C₄ alkoxy,C₂-C₄ alkanoyl, carbo(C₁-C₄ alkoxy), or amino(C₁-C₄ alkyl); R³ is phenylwhere the phenyl group is optionally substituted once withtrifluoromethyl or N-acetylamino; R⁴ is hydrogen, C₁-C₆ alkyl, or phenylwhere the phenyl group is optionally substituted once with nitro, C₁-C₄alkyl, trifluoromethyl, C₁-C₄ alkoxy, C₂-C₄ alkanoyl, carbo(C₁-C₄alkoxy), or amino(C₁-C₄ alkyl); R⁵ is hydrogen, C₁-C₄ alkyl, or phenyl;and R⁶ is phenyl or acetyl; or a pharmaceutical salt or solvate thereof;(b) one or more oncolytic agents; and (c) one or more pharmaceuticalcarriers, diluents, or excipients therefor.
 14. The formulationaccording to claim 13 where the oncolytic(s) is selected from:doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide. 15.The formulation according to claim 13 where the compound of formula I isa compound where R is COR¹ and R¹ is 3,4,5-trimethoxyphenyl.